Method of producing l-glutamic acid by fermentation of aromatic organic compounds

ABSTRACT

L-Glutamic acid is produced by culturing glutamic acid producing strains of Brevibacterium, Corynebacterium, Microbacterium, and Micrococcus on media which provide catechol, benzyl alcohol, benzoic acid, hydroxybenzoic acid, phenylacetic acid, protocatechuic acid, phenol and other aromatic compounds as at least 70 percent (by weight) of the carbon sources available during fermentation.

United States Patent Nishida et a]. Jan. 8, 1974 METHOD OF PRODUCING L-GLUTAMIC ACID BY FERMENTATION OF AROMATIC [52] US. Cl. 195/30, 195/49 ORGANIC COMPOUNDS [5]] Int. Cl CLZd 13/06 f h 8 4 [75] Inventors: Hiroshi Nishida, Yamato; Masao [58] Field 0 Scare 9 /2 3O 7 195/49 Yamamoto, Yatsush1ro; Atsuo Kltai, Kamakura; Asaichiro Ozaki, Tokyo; [56] References Cited Yukio Nishimura, Kamakura; Hiroto Takemi; Chimo Takemi, both of UNITED STATES PATENTS Yatsushiro, an f japan 3,511,752 5/1970 Tanaka et al. 195/28 R 3,450,599 6/1969 Tanaka et al. 195/30 [73] Assignees: Ajinomoto Co., Inc.; Sanraku Ocean (30-, 's Tokyo, Japan Primary ExaminerAlvin E. Tanenholtz [22 Filed: 8, 1972 Attorney-Kurt Kelman et al.

[21] Appl. No.: 232,950 57 ABSTRACT Related US, Application D t L-Glutamic acid is produced by culturing glutamic [63] continuatiomimpan of Sen 13 830 Fell 24 acid producing strains of Brevibacterium, Corynebac- 1970, abandoned. y terium, Microbacterium, and Micrococcus on media which provide catechol, benzyl alcohol, benzoic acid, 30 Foreign A fi ti priority Data hydroxybenzoic acid, phenylacetic acid, protocatechuic acid, phenol and other aromatic compounds as at least 70 percent (by weight) of the carbon sources available during fermentation.

8 Claims, No Drawings METHOD OF PRODUCING L-GLUTAMIC ACID BY FERMENTATION OF AROMATIC ORGANIC COMPOUNDS This application is a continuation-in-part of the copending application Ser. No. 13,830, filed on Feb. 24, 1970, and now abandoned.

This invention relates to the manufacture of L- glutamic acid, and particularly to a method of producing L-glutamic acid by fermentation.

L-Glutamic acid, hereinafter referred to as glutamic acid, has been produced heretofore by culturing suitable microorganisms on culture media which contain carbohydrates, acetic acid, ethanol, and aliphatic hydrocarbons as sole or principal carbon sources.

It has now been found that glutamic acid can also be produced by fermentation of culture media in which aromatic compounds provide most, at least 70 percent of the carbon available during fermentation.

Suitable compounds are benzoic acid, benzaldehyde, benzyl alcohol, resorcinol, catechol, phenol, mand phydroxybenzoic acid, gentisic acid, protocatechuic acid, 3,5-dihydroxybenzoic acid, phenylacetic acid, B-phenylpropionic acid, cinnamic acid, and their mixtures.

All the microorganisms of the genera Brevibacterium, Corynebacterium, Microbacterium, and Micrococcus which were known heretofore to produce glutamic acid by fermentation of other carbon sources have been found to metabolize at least several of the afore-mentioned aromatic compounds and to produce extracellular glutamic acid in good yields in culture media in which the aromatic compounds are the sources of at least 70 percent of the carbon available during fermentation.

The following microorganisms are preferably employed in the method of the invention:

Brevibacterium flavum ATCC 14067 Brevibacterium divaricatum NRRL 2311 Brevibacterium saccharolyticum ATCC 14066 Brevibacterium lactofermentum ATCC 13869 Brevibacterium aquapile ATCC 23587 Microbacterium ammoniaphilum ATCC 15354 Microcaccus glutamicus ATCC 13032 As is shown in Table l hereinbelow, these microorganisms are capable of producing economically significant amounts of glutamic acid in a culture medium in which one of the afore-mentioned aromatic compounds constitutes the sole significant source of carbon. This abil ity is shared by the following additional microorganisms:

Corynebacterium acetoacidophilum ATCC 13870 Corynebacterium acetoglutamicum ATCC 15806 and others.

The tests whose results are shown in Table l were performed by fermentation of aqueous culture media which, in addition to the listed carbon sources, contained 0.2 percent KH P 0.3 percent (NI-1.9 80 0.05 percent MgSO '7H O, 1.5 percent soybean protein hydrolyzate (Aji-eki), 3 7/1 biotin, 100 'y/1 thiamine hydrochloride, 2 ppm each Fe and Mn,

and had an initial pH of 6.8. All percentage values in this specification are by weight unless stated otherwise.

tion, and the carbon sources were replaced as they were being consumed. After 48 hours, the optical den sity (O.D.) of a specimen of each broth, diluted 20- fold, was measured at 610 mu, and the concentration of glutamic acid was measured by assay with Lactobacillus arabinosus.

The initial concentration of benzoic acid was 2.0 percent. Phenol, resorcinal, catechol, benzaldehyde, sodium 3,5-dihydroxybenzoate, sodium phenylacetate, sodium B-phenylpropionate, and sodium cinnamate were employed at an initial concentration of 0.2 percent, and the initial concentration of benzyl alcohol. sodium meta-hydroxybenzoate sodium parahydroxybenzoate, sodium gentisate, and sodium protocatechuate was 0.5 percent.

The total amount added was 1 percent for phenol; 2 percent for resorcinol, 3,5-dihydroxybenzoic acid, phenylacetic acid, B-phenylpropionic acid, and cinnamic acid; 3 percent for catechol; 6 to 10 percent for benzoic acid as indicated in the Table; and 4 percent for all other compounds. The strains of microorganisms are identified in the Table by their accession numbers of the American Type Culture Commission and the Northern Regional Research Laboratory of the US Department of Agriculture as indicated above.

Table 1 Carbon source Microorganism O.D. G.A.,g/dl

Phenol ATCC .380 0.3 23587 ATCC .305 0.3 13870 ATCC .255 0.3 13032 Benzoic acid, 6% ATCC .610 3.1

14066 8% ATCC .533 3.8

13869 8% ATCC .530 3.5

14067 8% NRRL 2311 .574 4.8 8% ATCC .605 4.3

23587 10% ATCC .583 3.3

13032 Benzoic acid, 8% ATCC .566 4.1

15354 8% ATCC .580 4.3

15806 i 6% ATCC .488 2.2

13870 Resorcinol ATCC .370 0.3

13870 ATCC .470 0.3

13032 Catcchol ATCC .540 0.5 23587 NRRL 23111 .490 0.7 ATCC .385 0.7

14067 ATCC .445 0.6

13869 ATCC .440 0.7

14066 ATCC .420 0.4

15354 ATCC .330 0.4

15806 ATCC .540 0.7

13870 ATCC .530 0.9

1 13032 Benzaldehyde ATCC .415 0.4

23587 NRRL 2311 .350 0.8 ATCC .490 0.7

14067 ATCC .520 1.3,

13869 ATCC .505 0.7

Table l-Continued Table l-Continued Carbon source Microorganism O.D. G.A.,g/dl on ou Microorganism 013- -.g/

ATCC .360 0,9 Phenyl acetic acid ATCC .420 0.4

15354 15806 H ATCC 35 Q2 ATCC .335 0.3

13370 13870 ATCC 430 1 5 ,B-Phenylpropionic acid .270 0.4

13032 Benzyl alcohol ATCC .400 1.2 23H 1 1 151512311 530 1 3 1467 ATCC 335 0 3 ATCC .480 1.2 3869 14067 ATCC .610 13 12;? 9 03 13869 H ATCC .285 0.3 fig 14066 H ATCC V295 05 15 x3225 .295 0.4

15354 B611z 1 61661161 ATCC .540 1.7 fig;

13870 ATCC 575 1'9 C1nnam1c ac1d .300 0.5 13032 Metahydroxybenzoic acid ATCC .365 1.5 23587 ATCC .225 0.4

NRRL 2311 .445 1.0 13869 ATCC .495 1.4 ATCC 390 0 7 4067 14066 i ATCC .465 1.4 ATCC 260 0 5 3869 13 870 l ATCC .340 0.8 ATCC 175 04 4066 13032 ATCC .430 1.5

15354 ATCC .380 1.3 u 88 490 I 3 A portlon of the aromatic compounds, not exceeding 3870 30 percent, may be replaced by more conventional car- ATCC .490 1.6 bon sources in the otherwise unchanged medium de- 13032 1 Parahydroxybenzoic acid ATCC .305 0.5 Scnbed above as 15 shown m Table 23587 NRRL 2311 .395 1.3 ATCC .440 1.5

14067 ATCC .425 1.5

131169 ATCC .415 1.3

14066 ATCC .430 1.4

15354 ATCC .310 1.6 40

15806 I ATCC .420 1.5 Tab 8 2 13870 ATCC Carbon sources Strain G.A., g/dl 13032 Gemisic acid ATCC W 3% 6a1661161+ 0.5% glucose ATCC 13032 1.4 23587 4% benlaldehyde 0.8% 116606 acid ATCC 13869 1.4 NRRL 2311 355 3% b611z 1 611661161 1% 11166s6 ATCC 23587 1.4 ATCC 410 LI 3% 06112 11 61661161 1% 6111111161 ATCC 14067 1.4 14067 5% benzoic acid 2% glucose ATCC 14066 4.8 ATCC .440 L1 6% benzoic acid 2% acetic acid ATCC 13869 5.3 13369 3% p-hydr0xybenzoic acid 1% ethanol NRRL 2311 1.8 1370 L2 3% protocatechuic acid 1% acetic acid ATCC 23587 1.8

14 ATCC .370 0.8 I 15354 ATCC .400 1.1

13870 ATCC .430 1.4

13032 Prntocatechuic acid ATCC .420 1.2

23587 NRRL 2311 .375 1.1 ATCC .450 1.8

14067 ATCC .455 1.8

13869 u ATCC .450 L6 All aromatlc compounds were present 1n1t1ally at a 14066 concentration of 0.3 percent and were replaced at 21 Eggs rate of 0.1 percent or 0.2 percent as they were being ATCC .420 1.5 consumed, the total amounts being listed in Table 2. u i gg 380 I 3 All glucose was added to the media at the start of fer- 13870 mentation, and a concentration of 0.5 percent ethanol fig? 5 lor acetic acid was originally supplied and replenished 3is mhydmxybenzoic acid ATCC .295 03 as needed. All aclds were added to the med1um 1n the form of their sodium or ammonium salts.

Biotin and compounds having biotin activity, such as biocytin, desthiobiotin, biotin sulfoxide, 7,8-diaminopelargonic acid.21-1Cl, and 7-keto-8-aminopelargonic acid.HCl; affected the growth of the microorganisms and the yield of glutamic acid as is shown in Tables 3 to 8, all conditions not stated otherwise being as de scribed with reference to Table 1, and glutamic acid (G.A.) yield being indicated in g/dl.

amounts suitably selected not to interfere with cell growth.

The aromatic compounds of the invention are employed at initial concentrations in which they are solui As is liri dwn to occur with more conventioiial carbon n Table 3 Biotin: 3 /1 5 /1 7 7/1 OD. GA. O.D. G.A. D. GA.

Bcnzoic acid 12% ATCC 13869 .72 4.75 .81 6.21 .90 6.03 Bcnzoic acid 10% ATCC 23587 .55 3.01 .63 3.82 .67 3.25 Bcnzoic acid 10% ATCC 15354 .70 4.23 .74 4.38 .78 4.56 Benzoic acid 12% ATCC 13870 .65 4.16 .78 5.87 .80 5.79 Bcnzoic acid 12% ATCC 13032 .68 4.18 .80 5.93 .85 6.02 p-Hydroxyb.a. 6% NRRL 2311 .47 3.06 .55 3.42 .61 3.18 p-Hydroxyb.a. 6% ATCC 23587 .36 1.20 .43 1.65 .49 1.92

' Table 4 Biocytin: 6 7/1 8 7/1 10 7/1 OD. GA. O.D. G.A. 0.1). G.A.

Bcnzoic acid 12% ATCC 14067 .70 4.57 .76 5.47 .78 5.21 Bcnzoic acid 12% ATCC 13032 .73 4.48 .80 6.02 .82 6.05 p-Hydroxyb.a. 8% ATCC 13869 .40 3.70 .52 4.12 .51 4.00 V p-Hydroxyb.a. 6% ATCC 13870 .54 2.53 .58 3.20 .61 3.21

W A """"''"fl Tab1e 5 Desthiobiotin: 6 7/1 8 7/1 10 -y/1 0.D. G.A., G.A. O.D. G.A.

Bcnzoic acid 10% NRLL 2311 .68 3.80 .80 4.32 .85 3.87 Benzoic acid 10% ATCC 15354 .69 4.03 .75 4.25 .78 4.32 Benzyl ale. 4% ATCC 14066 .44 0.95 .52 1.23 .60 1.25 Bcnzyl ale. 6% ATCC 13870 .53 2.01 .60 3.16 .62 3.03

- 1 M Table 6 Biotin sulfoxide: 4 W1 6 y/l 8 7/1 OD. G.A. 0.1). GA. G.A

Bcnzoic acid 10% ATCC 14066 .63 3.82 .70 4.38 .83 4.32 Bcnzoic acid 10% ATCC 15354 .65 3.65 .72 4.10 .79 4.16 m-l-lydroxyb.a. 8% ATCC 23587 .57 3.11 .61 3.67 .68 3.44 m-l-1ydroxyb.a. 8% ATCC 13032 .55 2.66 .60 3.78 .61 3.81

Table 7 7,8-Diaminopelarg.a c.2HCl 0.1 mg/l 0.2 mg/l O.D. GA. CD. 6.14. 0D. (LA.

Benzoic acid 12% ATCC 13869 .69 4.21 .84 5.85 .92 5.43 Benzoic acid 12% ATCC 13032 .70 4.35 .84 5.50 .88 5.00 Bcnzyl ale. 4% ATCC 14066 .45 1.27 .51 1.62 .55 1.09 Bcnzyl ale. 6% ATCC 13032 .51 2.55 .60 3.25 .68 2.31

.. o Table 8 7-Kcto-8-aminopcLacJ-1Cl 0.4 mg/l 6.775511" W n 0.8 mg 1 O.D. (LA. O.D. GA. CD. 6.14.

Benzoic acid 10% ATCC 23587 .62 3.25 .66 4.13 .69 3.72 Bcnzoic acid 10% ATCC 13870 .60 3.95 .67 4.30 .71 4.01 m'flydroxyba. 6% NRRL 2311 .46 2.22 .51 2.87 .56 2.76 m-Hydroxyb.a. 6% ATCC 15354 .49 2.61 .54 3.19 .60 2.95

1516 in the culture medium at the necessary p l -ll and it sources, surfactants such as polyoxyethylenesorbitan 65 is most convenient to add those which are acids in the monostearate, polyethyleneglycol monooleate, stearic acid, oleic acid, and the like can improve the yield of form of their sodium or ammonium salts. The amounts initially present in the medium are chosen so as not to inhibit cell growth and vary thus between 0.1 percent and 5.0 percent depending on the nature of the aromatic compound and the microorganism employed. If not the entire desired amount can be added to the medium initially, the aromatic compounds are replenished intermittently or continuously as they are being consumed.

A wide variety of suitable nitrogen sources is available and they include urea, aqueous ammonia, the ammonium salts or sulfuric, hydrochloric, carbonic, and nitric acid, and the entire amount of the nitrogenous material may be added at once to the medium in certain cases. However, a high concentration of ammonium ions enhances the inhibitory effect of the aromatic compounds on cell growth.

The necessary inorganic ions are provided by potassium phosphate, magnesium sulfate, small amounts of iron and manganese compounds, and the like. Organic growth promoters other than those described above include corn steep liquor, soybean protein hydrolyzate, bouillon, pepton, yeast extract, and other materials rich in organic nitrogen compounds.

An initial pH of approximately 7.2 is preferred, and a pH value of 6 9 is to be maintained during fermentation which is preferably carried out under aerobic conditions between 25 and 37 C. The glutamic acid is recovered from the culture broth in any conventional manner.

The following Examples further illustrate the method of the invention.

EXAMPLE 1 B. lactofermentum ATCC 13869 was inoculated in one liter of an aqueous culture medium containing 2.0 percent sodium p-hydroxy-benzoate, 0.3 percent ammonium sulfate, 0.1 percent KH PO. 0.1 percent K HPO 0.02 percent MgSO '7H O, 1.0 percent Ajieki, 5 ppm Fe, 5 ppm Mn, 2 y/] biotin, and 100 'y/l thiamine hydrochloride.

The aerobic culture was agitated at 30 C and kept at pH 7.8 by additions of ammonium hydroxide solution. 2 percent p-Hydroxy-benzoic acid in the form of its crystalline ammonium salt was added 6, 11, 1 6, arg again 20 hours after inoculation. The glutamic acid concentration in the medium reached 5.3 g/dl after 32 hours.

EXAMPLE 2 Sodium benzoate was substituted for the sodium phydroxy-benzoate in'the culture medium of Example 1, and the modified medium was inoculated with Cacetoacidophilum ATCC 13870. The cultivation was carried out aerobically with agitation at an initial temperature of 31 C. The temperature was lowered to 28 C during later stages when a 45 percent solution of sodium benzoate was supplied continuously at a rate to maintain a benzoic acid concentration of approximately 0.5 percent, and ammonium hydroxide solution to keep the pH at 7.9 while also providing assimilable nitrogen.

A total amount of 92 g benzoic acid was made available per liter of medium over 34 hours, and the glutamic acid formed amounted to 7.5 g/dl. The volume of the medium increased by 20 percent due to the additions made.

EXAMPLE 3 Microbacterium ammoniaphilum ATCC 15354 was inoculated on an aqueous medium containing 0.3 pe r cent benzyl alcohol, 0.1 percent ammonium sulfate, 0.2 percent urea, 0.1 percent KH PO. 0.05 percent MgSO '7H O, 1.5 percent Aji-eki, 2 ppm Fe, 2 ppm Mn, y/l thiamine hydrochloride, and 0.5 mg/l 7-keto-8-aminopelargonic acid hydrochloride.

After 18 hours of cultivation on a shaker at 30 C, 5 percent of the seed culture so prepared was inoculated in a fresh medium of the same composition, and the fermentation was carried out aerobically with agitation at 29 C. Benzyl alcohol was replenished 15 times at a rate of 0.3 percent during 40 hours according to its consumption, and the pH was held at 7.8 by adding ammonium hydroxide solution. The ultimate glutamic acid concentration was 1.93 g/dl.

EXAMPLE 4 In the medium of Example 1, the sodium phydroxybenzoate was replaced by 0.4 percent sodium protocatechuate, and the modified medium was inoculated with Microbacterium ammoniaphilum ATCC 15354. The culture was held on a shaker at 28 C, and sodium protocatechuate was fed at a rate of 0.4 percent eight times to replace the consumed compound. When the opticaldensity of a broth sample diluted to 20 times its volume reached 0.4, 0.05 percent polyoxyethylenesorbitan monostearate was added. The medium was kept at pH 7.5 by additions of a dilute ammonium hydroxide solution. After 48 hours, 2.4 g/dl glutamic acid had accumulated. in the medium.

EXAMPLE 5 An aqueous medium containing 2.0 percent sodium m-hydroxybenzoate, 0.3 percent ammonium sulfate, 0.1 percent KH PO. 0.1 percent K HPO. 0.02 percent MgSO '7H O, 3 ppm Fe, 3 ppm Mn, 1.0 percent corn steep liquor, and 100 341 thiamine hydrochloride was inoculated with Brevibacterium aquapile ATCC 23587, and the culture was agitated and aerated at 28 while the pH was held at 7.8 by additions of ammonium hydroxide solution. Crystalline ammonium mhydroxybenzoate equivalent to 2 percent mhydroxybenzoic acid was added after 6, 1 1, 16, and 20 hours to replace the consumed carbon source. After 32 hours of cultivation, the glutamic acid concentration in the medium reached 2.6 g/dl.

The broth (500 ml) was then centrifuged to remove the microbial cells, and the supernatant was partly evaporated to make it supersaturated with glutamic acid when its pH was adjusted to 3.2 with hydrochloric acid. The precipitated crude glutamic acid crystals were purified by carbon treatment, and 10.2 g L- glutamic acid sufficiently pure for conversion to monosodium glutamate was recovered.

c. recovering the accumulated L-glutamic acid, 3

l. at least 70 percent by weight of said source of assimilable carbon available in said medium during said culturing being constituted by at least one aromatic compound selected from the group consisting of benzoic acid, benzaldehyde, benzyl alcohol, resorcinol, catechol, phenol, metahydroxybenzoic acid, para-hydroxybenzoic acid, gentisic acid, protocatechuic acid, 3,5- dihydroxy-benzoic acid, phenylacetic acid, fi-phenylpropionic acid, and cinnamic acid.

2. A method as set forth in claim 1, wherein the initial concentration of said at least one aromatic compound in said medium is between 0.1 percent and 5.0 percent, and said medium is replenished with said aromatic compound as the latter is being consumed.

3. A method as set forth in claim 2, wherein a carbohydrate, acetic acid,- or ethanol constitutes an additional portion of said source of assimilable carbon.

4. A method as set forth in claim 2, wherein said medium contains a small amount of a substance having biotin activity sufficient to increase the amount of said accumulated L-glutamic acid.

5. A method as set forth in claim 2, wherein said at least one aromatic compound is benzoic acid.

6. A method as set forth in claim 1, wherein said at least one aromatic compound constitutes the sole significant source of carbon in said nutrient medium.

7. A method as set forth in claim 1, wherein said microorganism is capable of producing economically significant amounts of glutamic acid in a culture medium in which said at least one aromatic compound constitutes the sole significant source of carbon.

8. A method as set forth in claim 7, wherein said microorganism is a member of the group consisting of Brevibacterium flavum ATCC 14067, Brevibacterium divaricatum NRRL 231 1, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium lactofermentum ATCC 13869, Brevibacterium aquapile ATCC 23587, Microbacterium ammoniaphilum ATCC 15354, Micrococcus glutamicus ATCC 13032, Corynebacterium acetoacidophilum ATCC 13870, and Corynebacterium acetoglutamicum ATCC 15806. 

2. A method as set forth in claim 1, wherein the initial concentration of said at least one aromatic compound in said medium is between 0.1 percent and 5.0 percent, and said medium is replenished with said aromatic compound as the latter is being consumed.
 3. A method as set forth in claim 2, wherein a carbohydrate, acetic acid, or ethanol constitutes an additional portion of said source of assimilable carbon.
 4. A method as set forth in claim 2, wherein said medium contains a small amount of a substance having biotin activity sufficient to increase the amount of said accumulated L-glutamic acid.
 5. A method as set forth in claim 2, wherein said at least one aromatic compound is benzoic acid.
 6. A method as set forth in claim 1, wherein said at least one aromatic compound constitutes the sole significant source of carbon in said nutrient medium.
 7. A method as set forth in claim 1, wherein said microorganism is capable of producing economically significant amounts of glutamic acid in a culture medium in which said at least one aromatic compound constitutes the sole significant source of carbon.
 8. A method as set forth in claim 7, wherein said microorganism is a member of the group consisting of Brevibacterium flavum ATCC 14067, Brevibacterium divaricatum NRRL 2311, Brevibacterium saccharolyticum ATCC 14066, Brevibacterium lactofermentum ATCC 13869, Brevibacterium aquapile ATCC 23587, Microbacterium ammoniaphilum ATCC 15354, Micrococcus glutamicus ATCC 13032, Corynebacterium acetoacidophilum ATCC 13870, and Corynebacterium acetoglutamicum ATCC
 15806. 